SUMMARY Recent studies have revealed that the ?epitranscriptome,? i.e., the pattern and distribution of RNA modifications in mRNA, is dynamically regulated and has functional roles in the brain. Our laboratory had a founding role in the emergence of this field with our initial transcriptome-wide map of N6-methyladenosine (m6A), which revealed that m6A is present in over 8,000 different transcripts, and is predominantly located near stop codons and in the 5'UTR of mRNAs. Although m6A has been studied in standard tissue culture cell lines, the effects of m6A on mRNA in neurons remain unclear. Our data suggests that m6A may function as a cis-acting element that targets diverse mRNAs to dendrites. We also find that FMRP, a major RNA-binding protein linked to cognitive function, may bind a subset of m6A residues, i.e., the ones in the coding regions of mRNAs. These m6A sites show high overlap with known FMRP-binding sites in the transcriptome and depleting m6A from mRNAs markedly impairs the ability of FMRP to associate with translating ribosomes, which is believed to be essential for its translation-inhibitory effect. In order to significantly advance our understanding of these novel roles of m6A in neuronal function, the specific aims of this proposal are: (1) To image m6A mRNAs: Technologies for imaging m6A-mRNA trafficking in living cells and selective detection of m6A-mRNA in fixed cells. In this aim, we will develop two methods for imaging m6A-RNAs, one for live cells and one for fixed tissue. For live cell imaging, we will use a highly optimized MS2 system to image reporter RNAs that have been engineered to be resistant or susceptible to endogenous methylation. For fixed cell imaging, we have devised FISH-FRET, a fundamentally new approach for transcript- and m6A site-specific imaging of mRNA in virtually any cell or tissue. (2) To determine the role of m6A in mRNA trafficking and local translation. In this aim, we will test the role of m6A in mediating mRNA trafficking to axons and dendrites and how m6A influences local translation at these sites. (3) To determine if FMRP controls the function of mRNAs containing m6A in the coding sequence. In this aim, we will establish if FMRP's transcriptome-wide binding properties, granule formation and mobility in neurons are affected by m6A. We will also probe how FMRP controls translation and if its translation-inhibitory effect is directed towards mRNAs that contain m6A in their coding sequence. Although m6A is a highly abundant modification in neuronal mRNA, its functions remain unclear. Our studies point to key roles of m6A in directing mRNAs to dendrites and in mediating the assembly of FMRP onto stalled ribosomes and potentially FMRP granules. Our proposed studies have the potential to fundamentally alter our understanding of local translation, mRNA trafficking, FMRP function, and RNA regulatory mechanisms in the brain.